Establishment of spindle bipolarity is pivotal for proper chromosome segregation and mitotic progression. Mammalian polo-like kinase 1 (Plk1) has been thought to play a central role in this process. However, how Plk1 contributes to this event remains unknown. Here we demonstrated that Cdc2-dependent phosphorylation on a gamma-TuRC recruitment protein, Nedd1/GCP-WD, at two distinct sites provides both temporal and spatial cues to timely bring about Plk1 functions to the centrosomes and spindles via phosphorylation-dependent interaction with the polo-box domain of Plk1. At centrosomes, Plk1 interacted with a C-terminal phosphorylated motif of Nedd1 to phosphorylate the latter, a step suggested to be important for centrosome-based microtubule nucleation. Immediately following this event, Plk1 also interacted with an N-terminal phosphorylated motif of Nedd1 along the spindles and phosphorylated an Augmin subunit, Hice1, to promote microtubule-based microtubule nucleation. Loss of Nedd1-mediated Hice1 phosphorylation by Plk1 resulted in impaired Augmin interaction with microtubules and diminished gamma-tubulin recruitment to the spindles that ultimately led to defects in proper bipolar spindle formation and chromosome segregation. Taken together, the data provided here demonstrate the underlying mechanisms of how the two distinct Plk1-Nedd1 interactions at the centrosomes and spindles are deciphered into different biochemical and cellular outcomes to achieve normal bipolar spindle formation and mitotic progression. This study may allow us to understand the underlying mechanism of how Plk1 cooperates with other mitotic components to promote timely activation of specific protein complexes at distinct subcellular structures.